1. Technical Field
The present invention relates to a free radical initiator and a method for peptide sequencing using the same.
2. Background Art
Mass spectrometry has played a major role in opening up the new field of proteomics. Identification and characterization of a collection of proteins generally rely on gas-phase sequencing of peptides prepared by enzymatic proteolysis of proteins. Collisionally activated dissociation (CAD) of peptides has been the most widely used tool for sequencing. In recent years, electron capture dissociation (ECD) and electron transfer dissociation (ETD) have received extensive attention as an alternative tool, particularly useful for characterization of posttranslational modifications (PTMs) of proteins as well as for routine peptide analysis.
Top-down analysis of intact proteins by ECD is particularly appealing due to its promising capability for thorough survey of PTMs. In both ECD and ETD, odd-electron radical cation peptide species are prepared as a precursor to peptide backbone dissociations. In ECD and ETD, radical peptide species are formed by capturing an electron provided from a separate e-source or through collisions with anion species, respectively. Peptide backbone cleavage products in ECD/ETD are characterized with c/z ions as major products as well as with a/x ions as minor products, while b and y are main products of collision-based dissociation methods including CAD.
Alternatively, collisional activation of transition metal-peptide complexes has been shown to induce radical cation species. The subsequent collisional activation of the generated radical species followed ECD/ETD-like peptide dissociation pathways, giving rise to a, c, x, and z ions. Laskin et al. showed that charge-remote radical-driven fragmentation pathways are responsible for this type of peptide backbone dissociation (Anal. Chem. 2007, 79, 6607). Hydrogen abstraction by the radical site was suggested to initiate the subsequent backbone cleavages, which is also relevant to other odd-electron involving dissociation methods like ECD and ETD.
Another approach to introduce radical species is to attach a free radical initiator to peptide itself. Porter et al. converted N-terminal amines of lysine side chains to peroxycarbamates (J. Am. Chem. Soc. 2004, 126, 720; Med. Chem. 2006, 14, 6213; J. Am. Soc. Mass Spectrom. 2007, 18, 807). Collisional activation of peroxycarbamate peptide adducts complexed with Li, Na, K, or Ag ion gave rise to the neutral loss of the N-terminal side chain, i.e., radical species. Further fragmentation of the generated radical species showed an m/z shift of the b-ions corresponding to the loss of the N-terminal side chain. This shift labelled the b-ion series, which is useful for de novo analysis of peptides with the aid of SALSA algorithm. The Beauchamp group also investigated the use of free radical reactions as an tool for peptide and protein structure determination (J. Am. Chem. Soc. 2005, 127, 12436). They conjugated the water-soluble free radical initiator Vazo 68 to the N-terminus of a peptide. The MS/MS of the doubly protonated Vazo 68 conjugated peptides led to a free radical species generated by cleavage at the azo carbon. The product mass spectrum obtained by the subsequent CAD on the radical species showed a number of fragment ions, including many a and z ions, which are the signatures of odd-electron radical-driven fragmentation pathway. This approach was referred to as free radical initiated peptide sequencing (FRIPS).
In this patent application, we expand this FRIPS approach by employing a so-called ‘persistent radical’ precursor to the N-terminus amino groups or N-terminal amines of lysine side chains. The employed persistent radical species is ‘TEMPO (2,2,6,6-Tetramethylpiperidine-1-oxyl)’ which is a stable radical widely used as a free radical initiator in polymer chemistry as well as a structural probe in electron spin resonance spectroscopy (Scheme 1).

Compared with diazo or peroxy functionalized precursors, a TEMPO-based precursor of the present invention is chemically more robust and can generate radical species by homolytic cleavage upon thermal activation. In addition, the position of the radical site can be easily designated by placing a TEMPO group at the point of interest around the benzyl ring (see the molecular structure of 1 in Scheme 1).
The above information disclosed in this Background section is only for enhancement of understanding of the background of the invention and therefore it may contain information that does not form the prior art that is already known in this country to a person of ordinary skill in the art.
Throughout this application, several patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications is incorporated into this application in order to more fully describe this invention and the state of the art to which this invention pertains.